Why thaw embryos for PGT?
Preimplantation Genetic Testing (PGT) encompasses methods used to assess embryos produced through IVF for genetic abnormalities. These abnormalities may either impact the embryo's ability to successfully implant and result in a healthy pregnancy or can cause inherited genetic conditions in the offspring. PGT-A tests for aneuploidy, meaning an atypical number of chromosomes in the embryo. PGT-M tests for genetic variants in a specific gene that can cause an inherited genetic disorder (monogenic). PGT-SR screens for structural re-arrangements in chromosomes.
To obtain genetic material for testing, all types of PGT require a biopsy procedure. This involves removal of a small number of cells from the developing embryo, which are then sent to a specialized genetic testing laboratory for analysis. Some IVF clinics may have an in-house genetics lab, but most send out the biopsies to a specialized facility for genetic analysis. The biopsy almost always occurs in blastocyst stage embryos, and the cells removed during the biopsy are from the outer part of the embryo, called the trophectoderm.
In most cases, the embryos undergo biopsy during a fresh IVF cycle. This involves the collection of eggs, immediate fertilization, and subsequent biopsy after several days of culture (usually on Day 5). However, due to the typical timeframes associated with most PGT technologies, embryo transfer within the same fresh cycle is not feasible. It is typical for it to take a few weeks for PGT-A results to come back to the patient (usually longer for PGT-M and PGT-SR). Consequently, embryos are vitrified (frozen) shortly following biopsy and stored in liquid nitrogen. This freezing period allows for the genetic testing to be carried out, enabling patients to make informed decisions based on the genetic results. Based on the PGT results, they can then select which embryos to thaw for transfer at a later date.
Less commonly, patients may choose to biopsy previously frozen embryos to do PGT. In this scenario, embryos resulting from a prior egg retrieval have already been cultured and frozen. These previously cryopreserved embryos must be thawed for the embryologist to perform a trophectoderm biopsy and then refrozen on the same day. This means that the embryo will need to undergo an additional freeze-thaw cycle.
There are two main situations in which patients may choose to complete PGT of their frozen embryos, broadly categorized as: re-assessment of previously frozen embryos due to inconclusive PGT results and PGT for previously untested embryos.
Re-biopsy and Re-assessment of Previously Tested Embryos
Occasionally, embryos biopsied during the initial fresh cycle may yield inconclusive, incomplete, or failed genetic test results. When this occurs, patients may opt to retest these embryos to obtain definitive results. This is often the case if no other genetically normal (euploid) embryos are available for transfer and the patients/providers feel that having the PGT result is important, I.e., they do not want to transfer embryos with inconclusive results. Patients may alternatively decide to undergo another IVF cycle to produce more embryos or consider a blind transfer of the PGT-inconclusive embryo. Each of these choices carries its own set of advantages and risks.
Re-biopsy of embryos with mosaic results (with the aim of confirming chromosomal status) is discouraged by some professional organizations, such as the European Society for Human Reproduction and Embryology (ESHRE), due to a lack of diagnostic value and risk to the embryo.i In line with this, emerging evidence indicates that the transfer of mosaics should be considered (especially low level and certain single chromosome aneuploidies), which negates the need to thaw and retest.ii
Analysis of previously untested embryos
Patients who did not initially opt for PGT during the fresh cycle may later decide to pursue it. Reasons for this decision may include recurrent pregnancy loss (RPL), repeated implantation failure (RIF), advanced maternal age at the time of freezing, gender selection for family balancing (subject to ethical and legal considerations), the use of a gestational carrier, and/or the desire to select the best embryo when many frozen blastocysts are available. Additionally, patients may acquire new medical information, such as being carriers for specific genetic variations or structural rearrangements, prompting them to choose PGT-M or PGT-SR to avoid passing on genetic disorders.
How embryo thawing works
Vitrification is the method currently used for cryopreservation, as it is more efficient than the previous method of slow freezing. During thawing, frozen embryos are recovered to natural physiological temperature, restoring their biological activity. Various protocols and kits are available to IVF labs for this process. All protocols involve extracting embryos from the liquid nitrogen tank, warming them, and replacing the cryoprotectant solution used during freezing with water to rehydrate the cells.iii Cryoprotectants are crucial for safeguarding the embryos during freezing, as they help draw water out of the cells. Water is removed so that ice crystal formation during freezing is prevented, minimizing the risk for potential damage caused by ice crystals.
Embryo thawing is a relatively quick procedure performed by skilled embryologists within the IVF laboratory, typically taking less than 15 minutes. Once successfully thawed, embryos are transferred to a culture medium designed to support their viability, until subsequent biopsy by the embryologists.
Success rates when thawing embryos for genetic testing
The success of PGT for previously frozen embryos depends on the ability of the embryo to survive thaw, to avoid additional damage or stress from the additional freeze-thaw cycle, and the ability to achieve an accurate genetic diagnosis.
Thaw survival
Thaw survival rates for frozen embryos are generally high, with most embryos successfully enduring the thawing process. However, despite high thaw survival rates, this does not mean that re-biopsy and a second freeze-thaw cycle does not impact embryo quality. In addition, success rates can vary across different laboratories, depending on specific cryopreservation methods, technologies, and skills of the embryologists.iv
Vitrification, the gold standard technique for freezing, is associated with notably higher survival rates, typically above 95 percent.v This surpasses older slow freezing methods, which tend to yield survival rates of around 70 percent to 90 percent.vi The key advantage of vitrification lies in its rapid cooling process, minimizing ice crystal formation and potential damage to the cellular structure of the embryo.vii,viii,ix It is unlikely that any IVF labs are still using slow freezing methods for clinical practice.
Various factors, both in the lab and inherent in the biology of embryos, influence their chances of surviving vitrification. Lab-related factors involve the cryoprotectants used, which can be toxic to the embryos, as well as the speed at which embryos are cooled and warmed.x Studies have shown that the morphological quality of embryos and their developmental stage before freezing is linked to their ability to survive thawing.xi,xii,xiii A study of nearly 12 000 vitrified blastocyst-stage embryos found that thaw-survival rates were highest in embryos with lower rates of expansion or a highly scored trophectoderm (e.g., formed of many cells forming a cohesive, uniform layer).xiv This study also found that embryos frozen on day 5 had higher rates of survival than those frozen on day 6.
Genetic testing results
The ability to successfully obtain a genetic result from previously frozen (then thawed) embryos appears to be comparable to fresh embryos.xv,xvi Additionally, studies have been highly successful in obtaining genetic results from frozen embryos that previously yielded inconclusive results, and the probability that an embryo is genetically normal does not appear to be altered.xvii,xviii,xix,xx Promisingly, a multicenter study published in Human Reproduction examined more than 200 embryos that had initially yielded inconclusive PGT-A results. Among the embryos that survived the initial thaw (97 percent), 52 percent were confirmed to be genetically normal. Notably, some of these embryos resulted in successful live births when transferred to patients, underscoring the significant value of re-biopsying undiagnosed blastocysts in cases with initially inconclusive results.xxi
However, it is important to note that performing a biopsy at the cleavage stage (e.g., day 3 embryos) is associated with diminished clinical outcomes.xxii Therefore, it is highly recommended that frozen cleavage-stage embryos thawed for PGT should undergo further culture until the blastocyst stage before biopsy and subsequent refreezing. Using this approach, a study reported equivalent rates of live birth and miscarriage following the PGT analysis of frozen day 3 embryos that were thawed and cultured to blastocysts compared to embryos that were initially frozen as blastocysts.xxiii
Risks of an extra freeze-thaw cycle on embryos
The risks and benefits associated with subjecting previously tested frozen embryos to additional freeze-thaw cycles and biopsy procedures during preimplantation genetic testing (PGT) raises questions regarding safety and risk to the embryo, and to the subsequent pregnancy. It is essential to explore both the potential impacts of a second freeze-thaw cycle as well as a second freeze-thaw with a double-biopsy. These considerations are crucial for patients and healthcare providers in making informed decisions.
Risks: Analysis of previously untested frozen embryos
Performing PGT on cryopreserved embryos involves subjecting them to a second round of freezing and thawing, which introduces additional risks. Numerous studies have explored the impact of a second freeze-thaw cycle on embryos, with most reporting no significant differences in clinical outcomes (thaw survival, clinical pregnancy, live birth, or miscarriage rates) compared to embryos frozen only once.xxiv,xxv,xxvi,xxvii,xxviii Conversely, two further studies found that a second freeze-thaw cycle during the PGT analysis of frozen embryos was associated with reduced rates of thaw survivalxxix and live birth.xxx The negative impact of a second freeze-thaw cycle on live birth rates was also reported in statistical analysis of the data from several different studies.xxxi
These factors are especially important to consider as embryos labelled "mosaic“ or even "aneuploid” by PGT have resulted in healthy live births in some cases. In other words, abnormal PGT results may not always indicate an abnormal embryo with certainty, and couples should consider this when contemplating thawing of untested embryos for PGT biopsy. xxxii
Risks: Re-biopsy and analysis of previously tested frozen embryos
Additionally, patients considering PGT analysis of frozen embryos that were previously analyzed by PGT in the fresh cycle should also consider the potential impact of a second biopsy (re-biopsy) procedure, as embryo biopsies can carry inherent risks. However, the effect of the double-biopsy, double-freeze-thaw approach is not well-documented, and the current evidence is conflicting.
Several studies have investigated the impact of a double-biopsy, double-freeze-thaw approach on pregnancy rates in comparison to a single biopsy and freeze-thaw cycle, providing conflicting results. Three notable studies reported reduced pregnancy rates associated with the former approach when embryos previously analyzed were thawed for a re-biopsy. In particular, one of the largest studies, conducted by Neal et al. (2019), revealed a concerning 25 percent decrease in ongoing pregnancy rates in cases involving a second biopsy and freeze-thaw cycle, compared to embryos biopsied and frozen-thawed once.xxxiii Similarly, clinical pregnancy rates were found to be reduced in two further studies analyzing the impact of re-biopsy, highlighting the potential adverse impact that re-biopsy of previously tested frozen embryos can have on clinical outcomes.xxxiv,xxxv
Conversely, two promising studies have reported no detrimental effects on clinical outcomes when embryos undergo double biopsy and double freeze-thaw procedures.xxxvi,xxxvii Encouragingly, the studies demonstrated a 97-100 percent survival rate among embryos subjected to this approach during the second thaw. Furthermore, there were no observed adverse effects on implantation or pregnancy rates compared to embryos undergoing a single biopsy and freeze-thaw cycle. These findings suggest that a double-biopsy, double-freeze-thaw approach may be a viable option for patients without compromising embryo viability. Notably, the larger study conducted by Cimadomo et al (2018) reported live birth rates similar to those previously reported for embryos biopsied and frozen-thawed only once, reinforcing the potential benefits of this approach.xxxviii
A 2023 statistical analysis of data from multiple independent studies (meta-analysis) xxxix revealed that embryos subjected to re-biopsy and re-vitrification achieved comparable live birth and miscarriage rates to embryos biopsied and vitrified only once. However, it is crucial to note that this study also identified a negative impact of a double freeze-thaw cycle alone on live birth rates, as mentioned above, highlighting the conflicting nature of current evidence on this topic.
These variations in outcomes may arise from differences in laboratory protocols, cryopreservation techniques, and biopsy methods. For example, biopsy and PGT-A procedures lack standardization, and certain methods may be safer and more accurate than others, contributing to the variability in study results.xl,xli,xlii
While this field remains relatively underexplored, it is important to acknowledge that biopsy and cryopreservation procedures are invasive and can introduce additional risks to embryos.xliii Therefore, when possible, performing embryo biopsy for PGT during the fresh cycle may be advisable. However, considering the reasonable success rates reported in studies, PGT analysis of frozen embryos can be considered as an alternative to discarding existing embryos or undergoing another cycle of invasive ovarian stimulation.
Risks in women over 40
Women of an older age (e.g., over 40 years old) may face unique challenges and additional risks compared to younger patients. It is well-established that as women age, their ovarian reserve decreases, leading to fewer eggs being retrieved during an IVF cycle. Consequently, these women may have small numbers of embryos frozen in storage and any damage or loss during the biopsy or freeze-thaw procedures may further reduce the number available for transfer. Embryo quality and competence is also negatively associated with maternal age, and so it is possible that embryos obtained from older women may have a reduced ability to withstand multiple biopsies and freezing-thaw procedures.xliv
One reason that embryos may be more subject to the stress of an extra freeze-thaw cycle is due to alterations of mitochondria in oocytes and embryos from women of advanced maternal age. Oocytes from older women often exhibit decreased quality in terms of mitochondrial function, energy (‘ATP’) production, and antioxidant defenses. These factors may make the resulting embryos more susceptible to certain types of damage, such as oxidative stress and DNA damage, which may occur during cryopreservation.xlv,xlvi,xlvii,xlviii,xlix However, research in this area is very limited and unclear.
Another issue to consider is the cost-effectiveness of PGT, especially when it requires an additional thaw. Although there is relatively little evidence related to cost-benefit analysis, one Australian study examined the costs of PGT-A in 2093 women of advanced maternal age (>37yr) undergoing IVF. They found that the cost was over $28,000 for each additional live birth resulting from PGT.l
Conclusion
Performing PGT biopsy of embryos during the fresh cycle is advisable. However, some women may choose to do PGT later on cryopreserved embryos, for various reasons. This includes PGT for embryos that have not been previously tested, or those with inconclusive results. To complete the procedure, frozen embryos must be thawed to conduct a trophectoderm biopsy for the preimplantation genetic testing. This involves subjecting the frozen embryos to a second round of freezing and thawing, which may introduce additional risks. Furthermore, conducting a second biopsy on embryos with inconclusive results imparts additional risks beyond the second freeze-thaw cycle. However, considering the reasonable success rates reported in studies, PGT analysis of previously frozen and untested embryos is worth considering if the alternative would be discarding existing embryos or undergoing another cycle of invasive ovarian stimulation. Women should discuss the potential benefits and risks associated with thawing embryos for PGT or re-biopsy with their fertility specialist.
I Martine De Rycke, C., et al. (2022). ESHRE survey results and good practice recommendations on managing chromosomal mosaicism. Human Reproduction Open, 2022(4). https://doi.org/10.1093/hropen/hoac044
ii Viotti, M., et al. (2021). Using outcome data from one thousand mosaic embryo transfers to formulate an embryo ranking system for clinical use. Fertility and Sterility, 115(5), 1212–1224. https://doi.org/10.1016/j.fertnstert.2020.11.041
iii Edgar, D. H., & Gook, D. A. (2012). A critical appraisal of cryopreservation (slow cooling versus vitrification) of human oocytes and embryos. Human Reproduction Update, 18(5), 536–554. https://doi.org/10.1093/humupd/dms016
iv Kader, A., et al. (2009). Factors affecting the outcome of human blastocyst vitrification. Reproductive Biology and Endocrinology, 7(1). https://doi.org/10.1186/1477-7827-7-99
v Nagy, Z. P., et al. (2020). Vitrification of the human embryo: a more efficient and safer in vitro fertilization treatment. Fertility and Sterility, 113(2), 241–247. https://doi.org/10.1016/j.fertnstert.2019.12.009
vi Edgar, D. H., & Gook, D. A. (2012). A critical appraisal of cryopreservation (slow cooling versus vitrification) of human oocytes and embryos. Human Reproduction Update, 18(5), 536–554. https://doi.org/10.1093/humupd/dms016
vii Debrock, S., et al. (2015). Vitrification of cleavage stage day 3 embryos results in higher live birth rates than conventional slow freezing: a RCT. Human Reproduction, 30(8), 1820–1830. https://doi.org/10.1093/humrep/dev134
viii Rienzi, L., et al. (2016). Oocyte, embryo and blastocyst cryopreservation in ART: systematic review and meta-analysis comparing slow-freezing versus vitrification to produce evidence for the development of global guidance. Human Reproduction Update, 23(2), 139–155. https://doi.org/10.1093/humupd/dmw038
ix Nagy, Z. P., et al. (2020). Vitrification of the human embryo: a more efficient and safer in vitro fertilization treatment. Fertility and Sterility, 113(2), 241–247. https://doi.org/10.1016/j.fertnstert.2019.12.009
x Nagy, Z. P., et al. (2020). Vitrification of the human embryo: a more efficient and safer in vitro fertilization treatment. Fertility and Sterility, 113(2), 241–247. https://doi.org/10.1016/j.fertnstert.2019.12.009
xi Vanderzwalmen, P. (2003). Vitrification of human blastocysts with the Hemi-Straw carrier: application of assisted hatching after thawing. Human Reproduction, 18(7), 1504–1511. https://doi.org/10.1093/humrep/deg298
xii Cobo, A., et al. (2012). Outcomes of vitrified early cleavage-stage and blastocyst-stage embryos in a cryopreservation program: evaluation of 3,150 warming cycles. Fertility and Sterility, 98(5), 1138-1146.e1. https://doi.org/10.1016/j.fertnstert.2012.07.1107
xiii Coello, A., et al. (2021). Prediction of embryo survival and live birth rates after cryo-transfers of vitrified blastocysts according to morphology and day of blastulation: a retrospective study including 11936 blastocysts. Reproductive BioMedicine Online. https://doi.org/10.1016/j.rbmo.2021.02.013
xiv Coello, A., et al. (2021). Prediction of embryo survival and live birth rates after cryotransfers of vitrified blastocysts. Reproductive Biomedicine Online, 42(5), 881–891. https://doi.org/10.1016/j.rbmo.2021.02.013
xv Wilding, M., et al. (2019). Thaw, biopsy and refreeze strategy for PGT-A on previously cryopreserved embryos. Facts, Views & Vision in ObGyn, 11(3), 223–227. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7020945/
xvi Zhang, Q., et al. (2023). Impact of Multiple Vitrification-Warming Procedures and Insemination Methods on Pregnancy and Neonatal Outcomes in Preimplantation Genetic Testing for Aneuploidy. Reproductive Sciences (Thousand Oaks, Calif.), 30(7), 2302–2312. https://doi.org/10.1007/s43032-023-01177-0
xvii Cimadomo, D., et al. (2018). Inconclusive chromosomal assessment after blastocyst biopsy: prevalence, causative factors and outcomes after re-biopsy and re-vitrification. A multicenter experience. Human Reproduction, 33(10), 1839–1846. https://doi.org/10.1093/humrep/dey282
xviii Neal, S. A., et al. (2017). High relative deoxyribonucleic acid content of trophectoderm biopsy adversely affects pregnancy outcomes. Fertility and Sterility, 107(3), 731-736.e1. https://doi.org/10.1016/j.fertnstert.2016.11.013
xix Carles, M., et al. (2022). Second biopsy for embryos with inconclusive results after preimplantation genetic testing: Impact on pregnancy outcomes. Journal of Gynecology Obstetrics and Human Reproduction, 51(8), 102436–102436. https://doi.org/10.1016/j.jogoh.2022.102436
xx De Vos, A., et al. (2020). Multiple vitrification-warming and biopsy procedures on human embryos: clinical outcome and neonatal follow-up of children. Human Reproduction, 35(11), 2488–2496. https://doi.org/10.1093/humrep/deaa236
xxi Cimadomo, D., et al. (2018). Inconclusive chromosomal assessment after blastocyst biopsy: prevalence, causative factors and outcomes after re-biopsy and re-vitrification. A multicenter experience. Human Reproduction, 33(10), 1839–1846. https://doi.org/10.1093/humrep/dey282
xxii Scott, R. T., et al. (2013). Cleavage-stage biopsy significantly impairs human embryonic implantation potential while blastocyst biopsy does not: a randomized and paired clinical trial. Fertility and Sterility, 100(3), 624–630. https://doi.org/10.1016/j.fertnstert.2013.04.039
xxiii Theodorou, E., et al. (2022). Live birth rate following a euploid blastocyst transfer is not affected by double vitrification and warming at cleavage or blastocyst stage. Journal of Assisted Reproduction and Genetics. https://doi.org/10.1007/s10815-022-02440-0
xxiv Wang, X., et al. (2023). The effect of recryopreservation on embryo viability and outcomes of in vitro fertilization: a systematic review and meta-analysis. Fertility and Sterility, 120(2), 321–332. https://doi.org/10.1016/j.fertnstert.2023.03.001
xxv Wilding, M., et al. (2019). Thaw, biopsy and refreeze strategy for PGT-A on previously cryopreserved embryos. Facts, Views & Vision in ObGyn, 11(3), 223–227. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7020945/
xxvi Theodorou, E., et al. (2022). Live birth rate following a euploid blastocyst transfer is not affected by double vitrification and warming at cleavage or blastocyst stage. Journal of Assisted Reproduction and Genetics. https://doi.org/10.1007/s10815-022-02440-0
xxvii Zhang, Q., et al. (2023). Impact of Multiple Vitrification-Warming Procedures and Insemination Methods on Pregnancy and Neonatal Outcomes in Preimplantation Genetic Testing for Aneuploidy. Reproductive Sciences (Thousand Oaks, Calif.), 30(7), 2302–2312. https://doi.org/10.1007/s43032-023-01177-0
xxviii Bradley, C. K., et al. (2017). Impact of multiple blastocyst biopsy and vitrification-warming procedures on pregnancy outcomes. Fertility and Sterility, 108(6), 999–1006. https://doi.org/10.1016/j.fertnstert.2017.09.013
xxix Taylor, T. H., et al. (2014). Outcomes of blastocysts biopsied and vitrified once versus those cryopreserved twice for euploid blastocyst transfer. Reproductive Biomedicine Online, 29(1), 59–64. https://doi.org/10.1016/j.rbmo.2014.03.001
xxx Aluko, A., et al. (2018). Do multiple cryopreservation-warm cycles coupled with blastocyst biopsy impact IVF outcomes? Fertility and Sterility, 110(4), e88–e88. https://doi.org/10.1016/j.fertnstert.2018.07.265
xxxi Cimadomo, D., et al. (2018). Inconclusive chromosomal assessment after blastocyst biopsy: prevalence, causative factors and outcomes after re-biopsy and re-vitrification. A multicenter experience. Human Reproduction, 33(10), 1839–1846. https://doi.org/10.1093/humrep/dey282
xxxii Mastenbroek, S., et al. (2021). The Imperative of Responsible Innovation in Reproductive Medicine. The New England Journal of Medicine, 385(22), 2096–2100. https://doi.org/10.1056/nejmsb2101718
xxxiii Neal, S. A., et al. (2017). High relative deoxyribonucleic acid content of trophectoderm biopsy adversely affects pregnancy outcomes. Fertility and Sterility, 107(3), 731-736.e1. https://doi.org/10.1016/j.fertnstert.2016.11.013
xxxiv Bradley, C. K., et al. (2017). Impact of multiple blastocyst biopsy and vitrification-warming procedures on pregnancy outcomes. Fertility and Sterility, 108(6), 999–1006. https://doi.org/10.1016/j.fertnstert.2017.09.013
xxxv De Vos, A., et al. (2020). Multiple vitrification-warming and biopsy procedures on human embryos: clinical outcome and neonatal follow-up of children. Human Reproduction, 35(11), 2488–2496. https://doi.org/10.1093/humrep/deaa236
xxxvi Cimadomo, D., et al. (2018). Inconclusive chromosomal assessment after blastocyst biopsy: prevalence, causative factors and outcomes after re-biopsy and re-vitrification. A multicenter experience. Human Reproduction, 33(10), 1839–1846. https://doi.org/10.1093/humrep/dey282
xxxvii Carles, M., et al. (2022). Second biopsy for embryos with inconclusive results after preimplantation genetic testing: Impact on pregnancy outcomes. Journal of Gynecology Obstetrics and Human Reproduction, 51(8), 102436–102436. https://doi.org/10.1016/j.jogoh.2022.102436
xxxviii Cimadomo, D., et al. (2018). Inconclusive chromosomal assessment after blastocyst biopsy: prevalence, causative factors and outcomes after re-biopsy and re-vitrification. A multicenter experience. Human Reproduction, 33(10), 1839–1846. https://doi.org/10.1093/humrep/dey282
xxxix Cimadomo, D., et al. (2023). Opening the black box: why do euploid blastocysts fail to implant? A systematic review and meta-analysis. Human Reproduction Update, 29(5), 570–633. https://doi.org/10.1093/humupd/dmad010
xl Neal, S. A., et al. (2017). High relative deoxyribonucleic acid content of trophectoderm biopsy adversely affects pregnancy outcomes. Fertility and Sterility, 107(3), 731-736.e1. https://doi.org/10.1016/j.fertnstert.2016.11.013
xli Munné, S., et al. (2017). Euploidy rates in donor egg cycles significantly differ between fertility centers. Human Reproduction, 32(4), 743–749. https://doi.org/10.1093/humrep/dex031
xlii Bardos, J., et al. (2023). Reproductive genetics laboratory may impact euploid blastocyst and live birth rates: a comparison of 4 national laboratories’ PGT-A results from vitrified donor oocytes. Fertility and Sterility, 119(1), 29–35. https://doi.org/10.1016/j.fertnstert.2022.10.010
xliii Wei, Z., et al. (2021). Obstetric and neonatal outcomes of pregnancies resulting from preimplantation genetic testing: a systematic review and meta-analysis. Human Reproduction Update, 27(6), 989–1012. https://doi.org/10.1093/humupd/dmab027
xliv Cimadomo, D., et al. (2018). Inconclusive chromosomal assessment after blastocyst biopsy: prevalence, causative factors and outcomes after re-biopsy and re-vitrification. A multicenter experience. Human Reproduction, 33(10), 1839–1846. https://doi.org/10.1093/humrep/dey282
xlv Bentov, Y., et al. (2010). The use of mitochondrial nutrients to improve the outcome of infertility treatment in older patients. Fertility and Sterility, 93(1), 272–275. https://doi.org/10.1016/j.fertnstert.2009.07.988
xlvi Kader, A., et al. (2009). Evaluation of post-thaw DNA integrity of mouse blastocysts after ultrarapid and slow freezing. Fertility and Sterility, 91(5), 2087–2094. https://doi.org/10.1016/j.fertnstert.2008.04.049
xlvii Li, L., et al. (2011). Comparison of DNA apoptosis in mouse and human blastocysts after vitrification and slow freezing. Molecular Reproduction and Development, 79(3), 229–236. https://doi.org/10.1002/mrd.22018
xlviii Chiang, J. L., et al. (2020). Mitochondria in Ovarian Aging and Reproductive Longevity. Ageing Research Reviews, 63, 101168–101168. https://doi.org/10.1016/j.arr.2020.101168
xlix Liu, X., et al. (2021). The Feasibility of Antioxidants Avoiding Oxidative Damages from Reactive Oxygen Species in Cryopreservation. Frontiers in Chemistry, 9. https://doi.org/10.3389/fchem.2021.648684
l Lee, E., et al. (2019). A cost‐effectiveness analysis of preimplantation genetic testing for aneuploidy (PGT‐A) for up to three complete assisted reproductive technology cycles in women of advanced maternal age. Australian and New Zealand Journal of Obstetrics and Gynaecology, 59(4), 573–579. https://doi.org/10.1111/ajo.12988
